Research Journal of Biotechnology

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Vol. 10(10) October 2015

Genetic diversity of Coffea arabica in Ethiopia based on nuclear ribosomal DNA ITS sequences

Yan-Lin Sun, Byeong-Rok Yoo, Koo-Yeon Lee, Ho-Min Kang, Dong-Joo Lee, Tae-Hoon Choi, Cheng-Wu Jin*, Seon-Kang Choi and Soon-Kwan Hong*

Coffea arabica L. (arabica coffee) is originally indigenous to the mountains of the southwestern highlands in Ethiopia, representing the majority of the world’s coffee production and having a significant contribution to Ethiopia’s economy. The present paper was conducted to determine the genetic diversity of arabica coffee in Ethiopia for its conservation and breeding values based on the nucleonic ribosomal DNA internal transcribed spacer (nrDNA ITS) region sequence. Seventeen varieties were collected from 9 countries all around the world among which 8 represented varieties in Ethiopia.

Among the varieties, the highest diversity was recorded in the variety Ethiopia Hara Gold Bean (HG) and Ethiopia Sidamo (ES). High sequence variation also appeared among other varieties, suggesting that the genetic variation in arabica coffee was significant enough for good genetic conservation and breeding programs. This work not only provided more sequences of the nrDNA ITS region of C. arabica but also provided the basis for the clearer variety discrimination in Ethiopia.

Biosynthesis and Characterization of Gold Nanoparticles synthesized by Neosartorya udagawae

Jhansi Lakshmi V.* and Kannan K.P.

Gold nanoparticles (AuNPs) were synthesized at room temperature using Neosartorya udagawae fungal mat isolated from Indian Kolar Gold Field mine soil. Swift extracellular formation of AuNPs occurred within few seconds by the fungal mat incubated with 6mM concentration of tetra chloroauric (III) acid. The UV-Visible spectrum, Scanning Electron Microscopy and Transmission Electron Microscopy confirmed the presence of AuNPs in solution in the size range of 50-75 nm and showed most of the particles in spherical shape. Fourier Transform Infrared spectroscopy analysis showed that amino acids present in protein molecules on fungal mat surface were involved in reducing, capping and stabilization of synthesized AuNPs. X-ray diffraction revealed crystalline nature of bio reduced AuNPs. Toxicity study in animals showed almost nil toxicity. Fungal synthesized AuNPs were well tolerated and non-toxic.

A comprehensive approach of Botanical Compositions and Forage Yields in a Rangeland

Babalik Ahmet Alper* and Kilic Kamil

The aim of the study is to determine the plant-covered area, the botanical compositions and the forage yield in a natural rangeland. A case study was conducted of Daridere Watershed in Isparta district of Southern Turkey. Sampling was carried out in the north and south aspects in 2011. “Line intercept” and “quadrat” methods were used in order to determine the rangeland flora of the case study area. 190 plant taxa belonged to 41 families; out of which 36 taxa of Fabaceae were determined, while 21 and 17 taxa were determined in Caryophyllaceae and in Asteraceae respectively. The plant-covered area was found as nearly 27%. The botanical composition of rangelands’ taxa is approximately 48% Poaceae, 23% Fabaceae and 29% of other families. The dry forage and underground biomass yields were calculated as 368.76 kg/da and 545.03 kg/da respectively. The case study area of rangeland condition was established as “moderate”. Results of the study were also discussed based on rangeland management.

Design and Fabrication of Theophylline loaded tri-layer Nanostructure with magnetic core for Targeted Drug Delivery Application

Afsaneh Sharafi, Seyed Sadjadi Mirabdullah and Farhadyar Nazanin

In this work we report design and fabrication of theophylline loaded magnetic iron oxide nanostructure by adding appropriate amount of aqueous solution of theophylline in a dispersed solution of Fe3O4@SiO2@APTS @PEG conjugate dispersed solution of Fe3O4@SiO2@APTS @PEG conjugate prepared by microemulsion technique using water in n-hexane as water in oil phase. Hexadecyltrimethylammonium bromide (CTAB) and n-butanol were used as surfactant and cosurfactant.

Surface modification was carried out in pot using tetraethoxysilane (TESO), N-aminopropyltrimethoxy-silane (APTS) and polyethelenglycol (PEG). Characterization of the samples was carried out using X-ray powder diffraction (XRD), Scanning electron microscopy, X-ray energy dispersive techniques (SEM-EDX) and vibrating sample magnetometer (VSM) at room temperature. The loading of theophylline on nanostructured Fe3O4@SiO2@APTS @PEG conjugated with fast response to applied magnetic fields, zero remanence and coercivity properties was confirmed by Fourier transform infrared spectroscopy (FT-IR) and measured by UV spectroscopy.

Assessment of soil enzymes and PGP traits of rhizobacteria associated with rhizospheric soils of Indo Gangetic plains

Devi Shikha, Sharma Shivesh*, Tiwari Ashish and Singh Nand Kumar

The Indo Gangetic Plains (IGP) are one of the highly fertile and productive agricultural areas of the world, feeding millions of people. Challenges are there to maintaining soil health, increasing agricultural productivity and restoration of environmental quality by biological means major concerns in IGP region. Middle region of IGPs were selected for collection of soil samples and evaluated for assessment of bacterial diversity. A comparatively higher soil enzymes activity was recorded in most of rhizospheric soil samples as compared to non rhizosphere where activities are substantially lesser.

During the present study, almost 26.6% of bacterial diversity was found to be phosphate solubilizing and production of IAA was shown by majority of bacterial isolates approximately 89.1%. Siderophore production was detected in 12.5% of bacterial isolates. All the isolates were found to be catalase positive and HCN production was shown by only 16% of total bacterial isolates. The presence of multiple PGP traits in rhizospheric soil isolates suggested that these promising isolates can be used for development of microbial inoculants as a biofertilizer for enhancing the agricultural productivity, maintaining soil health and replacement of synthetic fertilizers in local agro climatic conditions of IGPs in India.

Oral administration of Tremella fuciformis conidium cells expressing glutamic acid decarboxylase improving the onset of type 1 diabetes in non-obese diabetic mice

ChungIl-Kyung and Park Hee-Sung*

The glutamic acid decarboxylase (GAD) gene construct under the control of the CaMV35S promoter was introduced into Tremella fuciformis yeast-like conidium(YLC) cells by Agrobacterium-mediated transformation method. Integration of GAD DNA into the YLC genome was confirmed by polymerase chain reaction (PCR) and dot blot hybridization and expression of the recombinant protein was verified by immunoblotting. Oral administration of GAD-expressing YLC cells in non-obese diabetic (NOD) mice improved the onset of diabetes by significantly reducing the level of blood glucose.

Synthesis of Hydroxyapatite over Keggin

Lakshmi Narsimhan G., Reddy Pradeep Vanga and Ashok M.*

Hydroxyapatite is a commonly known biocompatible and bioactive material employed in the field of medicine. Many of these properties depend on the particle size, grain size distribution and micro structural defects. Keggin is a mesoporous structured compound which can stack the biomolecules into it. We have successfully formed Hydroxyapatite by co-precipitation method over aluminium Keggin. The formation of the hydroxyapatite has been confirmed with XRD and the particle size has been calculated by using Scherrer formula. The organic bands have been studied with the FTIR bands and SEM images show the morphological properties of the Hydroxyapatite. EDX graph gives the chemical compositions.

The effects of UV-C on production of hypericin, phenolic and flavonoid in Hypericum triquetrifolium Turra

Toker Zuhal*, Karakaş Özgür and Özen Hasan Çetin

The current study was undertaken to determine the effects of UV-C radiation of total hypericin, phenolic and flavonoid content in Hypericum triquetrifolium Turra. The UV-C radiation was applied to H. triquetrifolium at different periods (15, 30, 45, 60 min). The highest total hypericin, phenolic and flavonoid accumulation 292±2.8, 357±3.4 and 331±3.7µgg-1 was achieved in 15 minutes of exposure to UV-C radiation when compared with the contents of control groups (140±2.3, 185±3.7, 173±2.5µgg-1).

Antiproliferative and antibacterial metabolites from endophytes isolated from Calotropis procera and Phoenix dactylifera

Abutaha Nael*, Al-Shami Muhammed, Semlali Abdelhabib, Baabbad Almohannad and Wadaan Muhammad A.

Endophytes associated with Calotropis procera and Phoenix dactylifera were screened for their capacity to produce bioactive metabolites against a panel of cancer cell lines and human pathogens. In the present study, the antiproliferative activity of ethyl acetate extract of MN05 displayed concentration-dependent inhibitory effects against the HepG2 and Jurkat cell lines, with an IC50 of 69.13 and 85.56µg/ml respectively. The extract induced morphological abnormalities in the cells and inhibited cells migration. Antimicrobial activity was found against both Gram positive and Gram negative microorganisms. No antifungal activity was detected against candida sp. MN05 extract showed very strong antibacterial effect against Mycobacterium sp. producing halos with an average diameter of 30.00±.0000 mm followed by Bacillus cereus (23.0±00 mm), Bacillus subtilis (20.05±0.21) and Proteus vulgaris (19.50±0.07). MN05 extract enhanced IL-8 and IL6 secretions in HepG2 cells. Further investigation to identify the active compound/s and mode of action is required.

Transcriptome-based reconstruction of gibberellic acid biosynthetic pathway in coconut (Cocos nucifera L.)

Shafeeq Rahman, Gangaraj K.P., Naganeeswaran S. and Rajesh M. K.*

Gibberellins are tetracyclic diterpenoid compounds which function as plant growth regulators and play an important role in many aspects of plant growth and development like stem elongation, seed germination and flower and fruit development. In the present study, we have carried out reconstruction ofGA biosynthetic pathway in coconut from the transcriptome data. Information about the reference genes involved in gibberellic acid biosynthesis was identified from KEGG pathway database. A total of seven such genes were identified in other model plants and corresponding sequences (gene/protein) were retrieved from Uniprot and NCBI-GenBank databases.

Based on these reference protein models, we have annotated all the seven GA biosynthesis genes (37 transcripts) in coconut leaf transcriptome using standalone Blast program. These transcripts were subjected to Gene Ontology (GO) search. Based on the results of the comparative analysis, GA biosynthetic pathway in coconut was reconstructed. The results of the present study can be used for further validation and isolation of full-length genes involved in gibberellic acid biosynthetic pathway in coconut.

Identification of specific inhibitors against Mycobacterium tuberculosis Thioredoxin C: Computational Study

Rohini Karunakaran* and Srikumar Padmalayam Sadanandan

The protein Thioredoxin C (Trx C) is a key regulator of cellular redox homeostasis and is responsible for the resistance in the pathogenic bacterium Mycobacterium tuberculosis (Mtb) and is one of the potential drug targets against tuberculosis to overcome the bacterial resistance. Hence, our computational work was focused on identification of specific inhibitors against Trx C using inhibitor PMX-464 derivatives as ligands dataset. Virtual screening method was implemented using Lipinski's rule of five and initial docking in Vina results in top six hits. Re-docking on hits in AutoDock, resulted in identification of two lead candidates with effective binding energy and weak interactions in the active site loop which favored the Trx C inhibition. In addition, molecular dynamics simulations results confirmed the affinity of lead compounds in dynamic system. In conclusion, the reported leads AGN-PC-0MUO8B and AGN-PC-0JRRZB are potential and specific inhibitors for Mtb Trx C and can act as future therapeutic agents for tuberculosis.

Microbial Dynamics of Polyhydroxyalkanoates Production from Waste Glycerol using RISA Technique

Kanchanasuta Suwimon and Pisutpaisal Nipon*

Ribosomal intergenic spacer analysis (RISA) technique was used to analyze the dynamics of microbial community capable of converting waste glycerol to polyhydroxyalkanoates (PHA) under cultivation on 5% v.v-1 crude glycerol as a carbon source. Three dominant band patterns were observed during 55 days cultivation. 16S rRNA sequences of dominant RISA bands showed bacterial community cultivated in waste glycerol were closely related to Azoarcus sp., Bacillus cereus, Bacillus pseudofirmus, Flavobacterium columnareand Thauera sp.The RISA technique could be used to identify dynamic microbial community in the bioreactor.

Advances in the research into transgenic rice expressing C4 photosynthesis enzymes

Zhang Bianjiang, Tang Ning and Hua Chun*

Due to the CO2 concentration mechanism, C4 plants exhibited higher photosynthetic efficiency than C3 plants under high light intensity, high temperature and high oxygen partial pressure conditions. During the last decade, studies on introducing C4 photosynthetic gene into C3 plants with genetic recombination technologies have drawn much attention. Such key genes of C4 photosynthetic enzymes have all been successfully introduced to C3 rice and have obtained some high expression strains. The point of new green revolution is being explored in the world. Constructing C4 rice based on good plant type is a reliable and effective approach to enhance photosynthetic efficiency in leaf. The physiological breeding of transgenic rice with C4 photosynthesis genes by a combination of conventional breeding and biotechnology would be one of the effective pathways in super-rice with high grain yield.

Molecular Cloning and Sequence Analysis of Novel Glutamate Decarboxylase Gene from Monascus Ruber Mr-5

Wu Qiaoyu, Qi Yuping, Feng Qingqing and Jiang Donghua*

A full-length cDNA encoding glutamate decarboxylase (designated as MrGAD) which catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA), was cloned from the GABA-rich Monacus ruber Mr-5 (CGMCC NO. M208043) using homology cloning and RACE. The cDNA of MrGAD has a 1536 bps open reading frame (ORF) encoding a protein of 511 amino acid residues. The deduced protein has an isoelectric point (pI) of 6.05 and a calculated molecular weight of 57.8 kDa. The amino acid sequence of MrGAD has more than 80% sequence identity with the GAD genes of some other fungi species as indicated by multiple alignments. Phylogenetic tree analysis reveals that MrGAD is more closely related to GADs from Aspergillus than to those of other fungi. The cloning and characterization of the MrGAD will enable us to enhance GABA production in Monacus ruber by improving the metabolic processes at the molecular level.

Isolation and identification of a new steroid degrading bacterial strain Raoultella ornithinolytica P718 from panda manure

Longyun Lin and Daren Pan*

Steroid contamination of environment is an ever growing problem and impacts population dynamics of human, animal and plant. In this work, we isolated a bacterial strain, P718, from panda manure which degraded steroids and was able to use steroids as carbon source. P718 was characterized as being gram negative and 16S rRNA analysis and API 50CH system test showed that it was Raoultella ornithinolytica belonging to the genus Raoultella the family Enterobacteriaceae. Strain P718 was able to grow in SIN medium either with testosterone or estradiol and estrone. These steroids also could be degraded in LB or SIN medium by strain P718. In our study, P718 was found to be more effective than Comamonas testosteroni ATCC 11996 for degrading testosterone, estradiol and estrone. Currently, work is ongoing to ﬁnd the steroid inducible proteins and genes.

Molecular cloning, characterization and expression pattern of a novel dihydroflavonol-4-reductase gene

Zeng Jianmin and Nian Fuzhao*

Anthocyanins have significant health benefits for animals and humans. Dihydroflavonol-4-reductase (DFR) has been characterized to a key enzyme in anthocyanin biosynthesis. The complete coding sequence of tobacco DFR gene was amplified by RT-PCR. The complete coding sequence of tobacco DFR gene was 990bp which encodes a protein of 329 amino acids. Sequence analysis revealed that the DFR of tobacco shares high homology with the DFR of wine grape (58%), soybean (58%), populus trichocarpa (56%) and barrel medic (56%). Phylogenetic tree analysis revealed that the tobacco DFR gene has a closer genetic relationship with that of soybean. Expression profile was studied and the results indicated that tobacco DFR gene was highly expressed in leaf and flower. These results established the primary foundation of utilizing tobacco anthocyanins as drugs for animals and humans in the future.

Micro-algae as a substrate for biofuel production – A review

Ranjitha J. and Vijayalakshmi S.*

This paper reviews the importance of micro-algae as a renewable sources of energy for the production of biofuels. Algal biomass can be ‘energy rich’, the growth of algae in dilute suspension at around 0.02-0.05 % dry solids possess considerable challenges in achieving a viable energy balance in micro-algal biofuel process operations. Algae-derived biofuel can reduce life cycle CO2 emissions by 50-70% compared to petroleum fuels. Algal biofuel is an alternative to fossil fuel that uses algae as its source of natural deposits. The energy crisis and the world food crisis have ignited interest in algaculture (farming algae) for making vegetable oil, biodiesel, biogasoline, biomethanol, biobutanol and other biofuels. Algae biofuels are biodegarable and relatively harmless to the enviroment.